marine drugs Article The Phytocomplex from Fucus vesiculosus and Ascophyllum nodosum Controls Postprandial Plasma Glucose Levels: An In Vitro and In Vivo Study in a Mouse Model of NASH Daniela Gabbia 1, Stefano Dall’Acqua 1, Iole Maria Di Gangi 2, Sara Bogialli 2, Valentina Caputi 1, Laura Albertoni 3, Ilaria Marsilio 1, Nicola Paccagnella 1, Maria Carrara 1, Maria Cecilia Giron 1 and Sara De Martin 1,* 1 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131 Padova, Italy; [email protected] (D.G.); [email protected] (S.D.A.); [email protected] (V.C.); [email protected] (I.M.); [email protected] (N.P.); [email protected] (M.C.); [email protected] (M.C.G.) 2 Department of Chemical Sciences, University of Padova, 35131 Padova, Italy; [email protected] (I.M.D.G.); [email protected] (S.B.) 3 Department of Medicine, General Pathology and Cytopathology Unit, University of Padova, 35128 Padova, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-049-827-5776; Fax: +39-049-827-5093 Academic Editor: Paul Long Received: 21 October 2016; Accepted: 9 February 2017; Published: 15 February 2017 Abstract: Edible seaweeds have been consumed by Asian coastal communities since ancient times. Fucus vesiculosus and Ascophyllum nodosum extracts have been traditionally used for the treatment of obesity and several gastrointestinal diseases. We evaluated the ability of extracts obtained from these algae to inhibit the digestive enzymes α-amylase and α-glucosidase in vitro, and control postprandial plasma glucose levels in a mouse model of non-alcoholic steatohepatitis (NASH); a liver disease often preceding the development of Type 2 diabetes (T2DM). This model was obtained by the administration of a high-fat diet. Our results demonstrate that these algae only delayed and reduced the peak of blood glucose (p < 0.05) in mice fed with normal diet, without changing the area under the blood glucose curve (AUC). In the model of NASH, the phytocomplex was able to reduce both the postprandial glycaemic peak, and the AUC. The administration of the extract in a diet particularly rich in fat is associated with a delay in carbohydrate digestion, but also with a decrease in its assimilation. In conclusion, our results indicate that this algal extract may be useful in the control of carbohydrate digestion and absorption. This effect may be therapeutically exploited to prevent the transition of NASH to T2DM. Keywords: Ascophyllum nodosum; Fucus vesiculosus; nonalcoholic steatohepatitis; postprandial blood glucose level 1. Introduction Edible seaweed, an easily available food source, has probably been consumed by coastal communities since the dawn of time, especially in Asia [1]. Seaweeds and their organic extracts are known to contain several bioactive polysaccharides with numerous health benefits [2–8]. These polysaccharides cannot be completely digested by the human digestive system and represent a source of dietary fiber, prebiotics, and other functional ingredients [9,10]. Soluble fiber can slow down the digestion and absorption of nutrients by increasing the viscosity, thereby decreasing blood sugar Mar. Drugs 2017, 15, 41; doi:10.3390/md15020041 www.mdpi.com/journal/marinedrugs Mar. Drugs 2017, 15, 41 2 of 12 levels and cholesterol [10]. Consistent with these observations, it has been reported that seaweed fiber consumption is associated with a significant reduction of chronic diseases, such as diabetes, obesity, and hypertension [11,12]. Recent studies have reported that fucoidan, a sulphated polysaccharide found in many species of brown algae and seaweed, has numerous biological and pharmacological activities, such as anti-proliferative, anti-inflammatory, and antiviral effects [13,14]. Algae extracts are considered a good source of digestive enzyme inhibitors. In particular, they contain polyphenolic compounds, such as bromophenols [15,16] and phlorotannins (PHTs) [5,17], which are well known α-glucosidase inhibitors. The enzyme α-glucosidase, together with α-amylase, is a key enzyme in starch breakdown and absorption. α-amylase is secreted from the pancreas and salivary glands [4], and catalyses the cleavage of α-D-(1-4) glycosidic linkages of starch, amylose, amylopectin, glycogen, and various maltodextrins, into shorter oligosaccharides, such as maltose and glucose [18,19]. α-glucosidase, which is also located in the brush-border surface membrane of intestinal cells, activates the final step of digestive processes by catalyzing the hydrolysis of complex carbohydrates and disaccharides to absorbable monosaccharides, e.g., the release of glucose from maltose and/or sucrose [20]. It has been reported that the inhibition of α-amylase and α-glucosidase can significantly lower the increase of the blood glucose level, after a mixed carbohydrate meal, by delaying the absorption of glucose [21]. In Type 2 Diabetes (T2DM) patients, this approach proves to be more efficient than controlling insulin secretion, since it is economic, convenient, and virtually prevents adverse drug reactions [22]. Nonalcoholic fatty liver disease (NAFLD) and its complication, nonalcoholic steatohepatitis (NASH), have become the most common causes of chronic liver disease in Western countries, causing considerable liver-related morbidity and mortality [23]. Evidence has accumulated that NAFLD may precede the development of T2DM [24], since one hallmark of this liver condition is insulin resistance, which is strongly associated with T2DM and abdominal obesity [24]. The subset of NAFLD patients who have NASH is estimated to be about 15% of the European [25] and USA [26] populations. Patients with NASH are at great risk of developing progressive liver disease and associated morbidity. Interestingly, the prevalence of NASH significantly increases in high-risk populations, since NASH is present in 25%–30% of patients with obesity or T2DM, and more than 35% of severely obese patients who have T2DM [27–29]. In this study, we analyzed in vitro inhibitory activity on the digestive enzymes α-amylase and α-glucosidase of, a commercially-available extract of Ascophyllum nodosum (egg wrack), and Fucus vesiculosus (bladder wrack). Following this, we studied its effect on blood glucose levels after starch ingestion in a mouse model of NASH, obtained by the administration of a high-fat diet (HFD) for five weeks [30,31]. Since NASH is often associated with insulin resistance and T2DM, the objective of this study was to ascertain whether seaweed extracts may be useful for glycaemic control in this liver condition. 2. Materials and Methods 2.1. Chemicals and Reagents Algae extract, commercially available under the trade name Gdue™ (Lot. N. 201400566), was provided by Aesculapius (Brescia, Italy). The extract was prepared from the dried thallus of Ascophyllum nodosum and Fucus vesiculosus, using a proprietary hot-water extraction process, followed by a series of filtration and ultrafiltration processes, and completed by spray-drying. Alginates and part of salts were removed during the process. The extract constituted a whole plant extract that contained plant polyphenols (35.5%, as indicated in the certificate of analysis of the commercial product), in addition to the algal polysaccharides (fibers) and minerals (iodine content <300 mg/kg). Starch potato, dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), sodium potassium tartrate, sodium hydroxide (NaOH), sodium chloride (NaCl), glutathione, human salivary α-amylase, a-glucosidase, acarbose, p-nitrophenyl-a-D-glucopyranoside (PNP-Gluc), and dinitrosalicylic acid (DNS), were obtained from Sigma Aldrich (St. Louis, MO, USA). Mar. Drugs 2017, 15, 41 3 of 12 2.2. NMR, HPLC-DAD, and GC-MS Analysis For fingerprinting of the extract, 1H-NMR spectra were acquired using a Bruker Avance III spectrometer (Billerica, MA, USA), operating at 400 MHz. The samples (100 mg) were weighted and extracted with 1 mL of solvent (deuterated methanol or deuterated water) in an ultrasonic bath, for 10 min. Samples were centrifuged (13,000 rpm) and the supernatant was transferred to an NMR tube to be measured. For the analysis of the polysaccharides, a Tosohaas PWXL 5000 9.4 × 300 mm was used as a stationary phase. An Evaporative Light Scattering Detector (Agilent, Santa Clara, CA, USA) was used to detect polysaccharides and a diode array detector was employed to observe absorption at 280 nm and the UV spectra. Elution was obtained using a mixture of 10 mM water and ammonium formiate with 1% of acetonitrile. The flow rate was set at 1 mL/min. Different dextrans (1000, 5000, 80,000, 150,000 Da) were used to create a calibration between the molecular weight and retention times. An estimation of the molecular weight of the polysaccharide fraction was obtained by comparing the retention times with the standard dextrans. GC-MS analysis of fatty acids was performed using a TRACE DSQ instrument (Thermo Scientific, Waltham, MA, USA), with a chromatographic column DB5 (30 m × 0.25 mm × 0.25 pm) and a helium gas flow rate of 1.2 mL/min. Chromatographic separation was performed using the following program: 100 ◦C for 4 min, an increase of 10 ◦C/min up to 200 ◦C, followed by an increase of 4 ◦C/min up to 260 ◦C. The algal sample was derivatized in order to detect the presence of the methyl esters of fatty acids, and to compare this with methylated standards. 2.3. a-Amylase Activity Assay A chromogenic 3,5-dinitrosalicylic acid (DNS) assay was employed to assess α-amylase activity, as described by Roy et al. [32]. Potato starch was solubilized by boiling a 1% solution for 15 min in 20 mM sodium phosphate (pH 6.9), containing 6.7 mM NaCl. The assay was carried out at 20 ◦C for 5 min in a final volume of 2.1 mL with α-amylase (0.83 µg/mL) and soluble starch (47.6 µg/mL). The inhibitory effect was measured by incubating 1 mL of substrate with 100 µL of algae extract solution at 20 ◦C for 10 min, prior to the assay.